Fig. 3
From: CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16
Promoter screening for heterotrophic and chemolithoautotrophic E. coli AppA production in C. necator H16 PHB-4. Cells were cultivated in mineral media containing kanamycin at 28 °C in baffled shake flasks till stationary phase. For heterotrophic growth, fructose was used as a carbon source. Chemolithoautotrophic cultivations were performed in an anaerobia pot under CO2/H2/O2 atmosphere. AppA expression levels with the different promoters were monitored by immunoblot with fructose (A) and CO2 (B) as carbon source. As a reference, the strong constitutive j5 promoter was used together with the C. necator derived promoters. Native promoters of the Calvin-Benson-Bassham operons (cbb_chr; cbb_pHG1), the membrane bound (mbh) and the soluble (sh) hydrogenases, a fructose degradation (frc) and a putative quorum sensing (phc) operon. As loading control, a Ponceau S staining of the membrane is shown. Phytase/phosphatase activity in the cell lysates (C) was determined by reactions with phytic acid as substrate (15 min, 37 °C) and a photometric measurement of the released phosphate. No background activity was found with an empty vector control (EVC)